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Keywords:

  • ascorbate peroxidase (APX);
  • catalase (CAT);
  • glutathione reductase (GR);
  • hydrogen peroxide (H2O2);
  • lipid hydroperoxides;
  • lipoxygenase (LOX);
  • Sinorhizobium–legume interaction;
  • superoxide dismutase (SOD)

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    The involvement of lipoxygenase and antioxidant enzyme activities as well as hydrogen peroxide (H2O2) accumulation are reported during early infection steps in alfalfa (Medicago sativa) roots inoculated either with a wild type Sinorhizobium meliloti or with a mutant defective in Nod-factor synthesis (Nod C).
  • • 
    Compatibility between M. sativa and Rhizobium correlates, at least in part, with an increase in the activities of these enzymes, particularly catalase and lipoxygenase, during the preinfection period (up to 12 h). The mutant strain, defective in Nod-factor biosynthesis, showed a decrease in all enzyme activities assayed, and an increase in H2O2 accumulation.
  • • 
    Enhancement of scavenging activities for several reactive oxygen species correlated with compatibility of the S. meliloti–alfalfa symbiosis, whereas the Nod C strain triggered a defence response. Nod factors were essential to suppress this response.
  • • 
    Increase in lipoxygenase and lipid hydroperoxide decomposing activities, observed during the first hours after inoculation with a compatible strain, could be related to tissue differentiation and/or the production of signal molecules involved in autoregulation of nodulation by the plant.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

All plants apparently exhibit the ability to distinguish between pathogenic and mutualistic interactions, since they readily enter into associations, but are simultaneously able to rebuff pathogenic challenges (Collinge et al., 1994). There may be similarities between pathogenic and mutualistic modes of infection and at least some elements of the molecular recognition mechanisms may be common in both cases. The end results of the recognition sequences are, however, very different. In pathogenesis, incompatibility leads to cell necrosis and cell death, whereby the mutualistic symbiosis is manifested by successful infection and nodule morphogenesis (Mellor & Collinge, 1995).

Rhizobium, as understood in general terms (Spaink, 2000), establishes an interesting mutualistic association with legumes by which bacteria provide plants with fixed nitrogen. In the Rhizobium–legume interaction, the plant plays an active role and exerts a strict control over nodulation events. Nod factor-induced developmental changes in early stages of mutualistic interaction share features of a plant defence response. Successful establishment of a symbiosis between Sinorhizobium and legumes requires either that the bacteria should not elicit a plant defence reaction or that they can elude it (Vasse et al., 1993). The idea that Nod factors may be described as elicitors comes from the observation that, like other well-known elicitors, they provoke alkalinization of the medium in tomato cell-culture systems (Staehelin et al., 1994).

Studies on incompatible plant–pathogen interactions have shown that salicylic acid (SA) is an endogenous signal involved in pathogenesis-related gene induction in plants (Malamy et al., 1996). We have previously found an accumulation of endogenous SA in alfalfa roots after inoculation with a mutant of Sinorhizobium meliloti blocked in Nod factor biosynthesis (Nod C), though not in roots inoculated with wild-type S. meliloti (Martínez-Abarca et al., 1998). Indeed, Nod factor-induced nodule formation was inhibited by exogenous SA. These results suggested that Nod factors could play an important role in blocking the plant defence response.

Several models have been proposed, describing the possible relationship between SA and the production of reactive oxygen species in the plant-hypersensitive response and in systemic acquired resistance (Ryals et al., 1996; Delaney, 1997). In this respect, the binding of SA to a protein having high homology with catalase (CAT) may lead to defence response induction through inhibition of this enzyme, resulting in accumulation of hydrogen peroxide (H2O2) and defence gene expression (Chen et al., 1993; Chamnongpol et al., 1998). Likewise, very high concentrations of H2O2 induce the production of SA (León et al., 1995). Moreover, biochemical studies have indicated that H2O2 removal is accomplished through a coupled series of redox reactions involving catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR), which is widespread in many plant organs, including N2-fixing root nodules (Dalton et al., 1993). Recently, Santos et al. (2001) reported production of H2O2 in 6-wk-old nodules of alfalfa infected with S. meliloti.

On the other hand, there are several reports of an earlier and greater induction of lipoxygenase (LOX) activity in the reactions of pathogen resistance (Croft et al., 1990). A role for the involvement of LOX in the development of the nodule has been proposed (Wisniewski et al., 1999). Recently, the expression of a Phaseolus vulgaris LOX (PvLOX5) has been described during nodule development and in roots inoculated with R. tropici, implying a role for this gene during symbiosis (Porta & Rocha-Sosa, 2000). In addition, it is known that the lipid hydroperoxides formed by the action of LOX upon polyunsaturated fatty acids are further metabolized and produce molecules such as jasmonic acid, which have been proposed to serve as mediators of the plant defence response to pathogen attack (Gundlach et al., 1992).

The aim of the present work has been to study comparatively the effects of inoculation of alfalfa (Medicago sativa) plants either with a wild-type strain of S. meliloti or with a mutant defective in Nod factor synthesis (Nod C). Specifically, the time-course and magnitude of root LOX, CAT and other antioxidant enzyme activities, and of H2O2 accumulation have been investigated during the early steps of interaction.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plant growth conditions and bacterial strains

Axenic cultures of alfalfa (Medicago sativa ecotype Aragón) plants were prepared in test tubes as previously described (Olivares et al., 1980). Plants (10-d-old) were inoculated with 108 cells ml−1 from a 24-h culture, prepared in tryptone-yeast extract medium, of a wild-type S. meliloti strain AK 631 or its Nod C derivative mutant (AK 1672) blocked in Nod factor biosynthesis (Kondorosi et al., 1984). Control plants (noninoculated) were grown with basal medium. Experiments were performed 4, 8, 12 and 24 h after inoculation. Roots of 50–100 plants from each treatment were frozen in liquid nitrogen and stored at −70°C for subsequent determination of enzyme activities.

Root protein extraction

Frozen roots were ground in a mortar with five volumes of 50 mM potassium phosphate buffer, pH 6.5, containing 1 mM EDTA, 6% (w/v) polyvinylpolypyrrolidone (PVPP), 1 mM dithiothreitol (DTT), and 1 mM phenylmethylsulphonyl fluoride (PMSF). After centrifugation (12 000 g, 20 min) the supernatant was collected and 75% (v/v) of cold acetone added with gentle stirring. Centrifugation was repeated and the pellet was recovered and resuspended in 1 ml of 50 mM potassium phosphate buffer pH 6.5, except for ascorbate peroxidase determination where 1 mM ascorbic acid was added to the buffer.

Enzyme assays

Lipoxygenase (LOX; EC 1.13.11.12) activity was assayed spectrophotometrically at 234 nm in a Shimadzu UV-160 spectrophotometer according to Axelrod et al. (1981).

Superoxide dismutase (SOD; EC 1.15.1.1) activity was determined by the ferricytochrome c method using xanthine/xanthine oxidase as the source of superoxide radicals, and a unit of activity was defined according to McCord & Fridovich (1969).

Catalase (CAT; EC 1.11.1.6) activity was determined by spectrophotometry at 240 nm according to Aebi (1984). Ascorbate peroxidase (APX; EC 1.11.1.11) was determined following the method described by Amako et al. (1994), and glutathione reductase (GR; EC 1.6.4.2) was determined according to Kang et al. (1996).

Preparation of lipid hydroperoxides

Lipid hydroperoxides were prepared using commercial α-linolenic acid and soybean lipoxygenase (LOX1, Sigma-Aldrich, Madrid, Spain) at its optimum pH of 9.0. Twenty units of LOX1 were added to 70 ml of 2 mM α-linoleate containing 0.2 M borate buffer, pH 9.0. The lipoxygenase reaction mixture was kept on ice under an atmosphere of pure O2. After incubation for 15 min with shaking, the reaction was stopped by acidification to pH 3.0 with 4 M HCl, and the products were extracted with an octadecyl solid-phase extraction column (C-18, 0.5 g) eluted with 2 ml of methanol. Then, the solvent was evaporated, the residue dissolved in 0.5 ml of methanol and subject to TLC on a 10 × 20 cm silica gel plate (DC-60, Merck), with n-hexane : diethyl ether : acetic acid (50 : 50 : 1; by vol). After running, the products were visualized under UV light, the hydroperoxide fractions were scraped off the silica gel plates and the gel was extracted with chloroform : methanol (2 : 1; by vol). After removal of silica gel, the amount of the purified α-linolenic hydroperoxides (LAOOH) was calculated assuming a molar absorbance of 25 000 M−1 cm−1 at 234 nm (Verhagen et al., 1977). The purified LAOOHs were stored as a solution in methanol at −20°C.

Assay of hydroperoxide-decomposing activity

Hydroperoxide-decomposing activity was measured in terms of the decrease in conjugated dienes, as followed by spectrophotometry at 234 nm, using LAOOHs as substrate. Before the assay, 0.03% (v/v) Tween-20 was added and the pH was adjusted to between 8 and 8.5 with 1 M NaOH to ensure substrate solubility. Initial rates of hydroperoxide-decomposing activity were monitored over 15 s at 25°C. One unit of activity was defined as the amount of enzyme that causes the disappearance of 1 µmol of hydroperoxide min−1 at 25°C.

Measurement of hydrogen peroxide

The method used was based on that of Okuda et al. (1991). Alfalfa roots were ground with mortar and pestle at 0°C for 1 min in the presence of 2 ml of 0.2 M HClO4 and 0.1 g of sea sand. The slurry was centrifuged at 20 000 g for 5 min. To remove HClO4, the supernatant was neutralized to pH 7.5 with 4 M KOH and the solution was centrifuged at 1000 g for 1 min. An aliquot (200 µl) of supernatant was applied to a 1-ml column of DEAE-Sephadex A-25 (0.8 cm × 2 cm), and the column was washed with 0.8 ml of distilled water. The eluate was used for the assay of H2O2.

The reaction mixture contained 1 ml of eluate, 400 µl of 12.5 mM (3-dimethylamino) benzoic acid (DMAB) in 0.375 M phosphate buffer (pH 6.5), 80 µl of 3-methyl-2-benzothiazoline hydrazone (MBTH) and 20 µl of peroxidase (0.25 unit) in a total volume of 1.5 ml. The reaction was started by addition of peroxidase at 25°C. The increase in absorbance at 590 nm was monitored in a Shimadzu UV-160 spectrophotometer.

Protein assay

Protein content was determined by the method of Lowry et al. (1951), using BSA (fraction V) as a standard.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

LOX activity was three- to fourfold higher in roots inoculated with the wild-type strain than in those inoculated with the Nod C mutant (Fig. 1). Hydroperoxide decomposing activity was detected in root protein extracts using LAOOHs as substrate. Since the decomposing activity was saturated before the substrate was completely consumed, the activity was measured in terms of the initial velocity of the reaction. The activity was higher in protein extracts from alfalfa roots inoculated with the wild-type strain than in those from roots inoculated with the mutant Nod C bacteria (Fig. 2).

image

Figure 1. Time course of lipoxygenase (LOX) activity in alfalfa roots inoculated with wild-type Sinorhizobium meliloti (closed circles) or with a mutant defective in Nod factor synthesis (open circles). Values in uninoculated roots are represented by open squares. Data show the mean of four independent experiments.

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image

Figure 2. Time course of hydroperoxide-decomposing activity of protein extracts from alfalfa roots inoculated with wild-type Sinorhizobium meliloti (closed circles) or with a mutant defective in Nod factor synthesis (open circles). Values in uninoculated roots are represented by open squares. Data show the mean of four independent experiments.

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Although SOD activity decreased below control levels in all cases, this was less pronounced when plants were inoculated with the wild-type strain. Similarly, the activities of CAT, APX, and GR decreased significantly below plant control levels in roots inoculated with the Nod C bacteria (Fig. 3). However, the activities of these enzymes increased to well above control levels in roots inoculated with the wild-type strain. The pattern of GR and, above all, CAT and APX were completely different in both strains of bacteria between 4 and 24 h after inoculation. Activities of CAT and APX peaked at 4 and 8 h after inoculation, returning to control levels at 24 h. Thus, it seems that the first 24 h of plant–bacteria interaction are critical for determining the plant response to compatible or incompatible bacteria.

image

Figure 3. Time course of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) activities in alfalfa roots inoculated with Sinorhizobium meliloti wild-type (closed circles) or with a mutant defective in Nod factor synthesis (open circles). Values in uninoculated roots are represented by open squares. Data show the mean of four independent experiments.

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In addition, and as could be expected, the accumulation of H2O2 also showed a different pattern in compatible as opposed to incompatible interactions (Fig. 4). While H2O2 was enhanced moderately in alfalfa roots inoculated with wild-type bacteria, a significant increase was detected in plants inoculated with the Nod C mutant.

image

Figure 4. Time course of hydrogen peroxide accumulation in alfalfa roots inoculated with wild-type Sinorhizobium meliloti (closed circles) or with a mutant defective in Nod factor synthesis (open circles). Values in uninoculated roots are represented by open squares. Data show the mean of four independent experiments.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Although the early steps of the interaction between host and microsymbiont bacteria have been well studied at the histological and ultrastructural levels, not too much is known about plant root cell function at this stage. Early infection steps in the compatible association established between legumes and Rhizobium could resemble a pathogen attack, although the outcome of the interaction is clearly different (Vance, 1983; Djordjevic et al., 1987). It is well known that the early stages of this process, including gene expression in the bacterium and cell growth, division, and differentiation in the host, are mediated by signal exchange between the host and symbiont (Spaink, 2000).

In the present report, attention has been focused on the relationship between LOX and antioxidant enzyme activities, as well as on hydroperoxide-decomposing activity and H2O2 accumulation during the early stages of establishment of the S. meliloti-alfalfa symbiosis in plants inoculated either with a wild-type strain of Sinorhizobium or with its Nod C derivative. The pattern of assayed enzyme activities and of H2O2 generation during root infection (Figs 1, 3, 4), suggest that compatibility of Rhizobium–alfalfa interaction depends, at least in part, on an increase in these activities during the preinfection period.

It has been shown that after the first nodules have formed following the inoculation of alfalfa by wild-type S. meliloti, the plant reacts to infection by displaying the cytological and biochemical features of a hypersensitive response (Vasse et al., 1993). The fact that nodulation is autoregulated indicates that mechanisms involved in control of nodulation are elicited during the symbiotic relationship. Existing evidence shows that, during the infection process, a high percentage of infection threads abort before a nodule is formed and the first nodules formed inhibit subsequent infection (Caetano-Anollés & Gresshoff, 1991). A rapid increase in LOX activity and/or mRNA in response to pathogen interaction has been reported in plants (Melan et al., 1993; Slusarenko, 1996) and may contribute to the resistance response through the production of signal molecules such as jasmonic acid. This molecule is recognized as one of the intracellular second messengers mediating a variety of cell responses, some of which are related to defence in many plant–pathogen interactions (Dixon et al., 1994; Hammond-Kosack & Jones, 1996). The expression pattern of four LOXs analysed in inoculated and uninoculated Phaseolus vulgaris roots revealed several differences (Porta & Rocha-Sosa, 2000). It is quite clear that during symbiosis the expression of one of these (Pv LOX5) was not only regulated in the nodule but in the root tissue as well. This regulation may be associated with cell growth and expansion and with a defence role. Our findings concerning the interaction between alfalfa and wild-type S. meliloti, suggest that an early event after bacteria recognition could be the activation of a LOX able to form fatty acid hydroperoxides that are further degraded in alfalfa root cells (Fig. 2). Metabolism of hydroperoxides produced by LOX activity could lead to compounds, such as jasmonic acid, with regulatory activities.

On the other hand, SA has been implicated as one of the key components in the signal transduction pathway leading to plant resistance to various pathogens (Ryals et al., 1996). In a previous report, we showed that inoculation of alfalfa roots with a strain of S. meliloti blocked in Nod factor synthesis (Nod C) lead to an increase in endogenous SA that was detectable 4 h after inoculation. By contrast, in alfalfa roots inoculated with the wild-type strain, SA remained at its basal level of activity (Martínez-Abarca et al., 1998). Such results suggested the involvement of Nod factors in the inhibition of SA-mediated resistance in legumes. Moreover, the detected enhancement in SA could lead to an inhibition of antioxidant enzyme activities, particularly CAT and APX, as well as of LOX activity (Figs 1, 3). In agreement with this, the pattern of all these enzyme activities is negatively correlated with the detected levels of SA in alfalfa roots (Martínez-Abarca et al., 1998), whilst endogenous concentrations of H2O2 were significantly enhanced in roots inoculated with the Nod C mutant (Fig. 4). In this respect, it must be kept in mind that SA has been considered as an inhibitor of H2O2 metabolism by blocking the activities of CAT (Chen et al., 1993) and APX (Durner & Klessig, 1995; Durner et al., 1997). Moreover, as León et al. (1995) reported, very high concentrations of H2O2 induce production of SA.

Since infection of roots with a mutant defective in Nod factor synthesis could trigger a defence response (salicylic acid accumulation), we can conclude from the present results that plant defence mechanisms are blocked during the compatible Rhizobium–legume symbiosis. The picture that emerges from these results allows us to propose that the high LOX and antioxidant enzyme activities, as well as hydroperoxide decomposing activity, detected in roots inoculated with the wild-type strain could be involved in the control of the oxidative burst generated during early steps of symbiosis and in the production of signal molecules by which plants autoregulate nodulation.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors wish to thank Mr Manuel Martínez for technical drawing of figures. This work has been supported by the Plan Andaluz de Investigación (PAI) (Junta de Andalucía, Spain).

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  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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